Study of radiotherapy resistance associated to EGFR/MAPK signaling pathway and its application in the rational design of a treatment with simvastatin in combination with cetuximab and radiotherapy (in experimental models of carcinoma)

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Study of radiotherapy resistance associated to EGFR/MAPK signaling pathway and its application in the rational design of a treatment with simvastatin in combination with cetuximab and radiotherapy (in experimental models of carcinoma)

Resum:

1. Generation and characterization of a radioresistant cell line
In the first part of this project of Thesis, we subjected culture cells to fractionated radiation and clonal selection, to obtain a modified cohort of cells. The aim of this study was to develop an isogenic resistant cell line which could be used to identify molecular changes associated with acquired resistance to radiation and tumour aggressiveness in cancer.
We developed the stable isogenic resistant A431-R cell line from parental A431 cells presented a diminution of the radiosensitivity (decreases in SF2 and α-component of LQ model). These changes were associated with an increased efficiency in DNA repair. We demonstrated that A431-R cells acquired higher cloning efficiency and faster growth and migration ability; these properties were observed to be associated with remarkable baseline levels of relevant oncoproteins and elevated angiogenic capabilities.
2. Development of a technique to irradiate mice bearing xenografts
In the second part of this project of Thesis, we have reached four objectives during the design procedure described to administer fractionated radiotherapy to immunodeficient mice in the Radiotherapy Department of a Hospital. First, we have minimized irradiation of healthy tissues around the tumour. Second, we have fulfilled the standard irradiation principles for human treatments. Third, we reduced time and increase efficiency by irradiating several mice at a time. Finally, our procedure was easy to reproduce and repeat on a daily basis by only two people. We demonstrated the feasibility of fractionated irradiation using immunodeficient mice to evaluate the role of radiotherapy on experimental tumours simulating a clinical setting.
3. Simvastatin sensitizes to radiotherapy plus cetuximab
In the third part of this project of Thesis, we pre-clinically explored whether a treatment regime involving the addition of simvastatin to XRT and C225 merits further research. We have shown that the addition of simvastatin significantly decreased proliferation and clonogenic survival of cells treated with XRT and C225. Moreover, we used an experimental model with tumor cells derived from squamous cell carcinoma of that suggests that simvastatin may increase the antitumor effect of XRT plus C225— at doses and fractions of XRT that mimic doses administered in the clinical setting.
The addition of simvastatin was associated with an increase in apoptosis and a decrease in the levels of activated ERK1/2, AKT, and STAT3 oncoproteins, a set of observations that provide support to the higher anti-tumor effects produced by the triple treatment.
An important consideration is that when we combined simvastatin with XRT + C225 in the treatment of the radioresistant generated A431-R, we observed that the initial resistance of these cells to the treatment is in part reverted. In fact, cell migration, cell proliferation and cell survival were decreased when simvastatin was combined to the treatment with XRT+C225.
In addition, tumour growth was slowed down when simvastatin was added to XRT+C225 treatment. The model A431-WT/A431-R we have generated can be a useful tool to examine new treatments directed to revert radioresistance produced from a prior treatment with radiotherapy. Treatment effects can be studied in both cell sublines and comparison with the parental cells could give important informatio